Amplification with primers of limited nucleotide composition
Inventors
Wang, Youxiang • Yang, Zhijie • Chen, Xin
Assignees
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Publication Number
US-11091799-B2
Publication Date
2021-08-17
Expiration Date
Abstract
The invention provides methods of amplification from a single primer or a pair of forward and reverse primers of limited nucleotide composition. Limited nucleotide composition means that the primers are underrepresented in at least one nucleotide type. Such primers have much reduced capacity to prime from each other or to extend initiated by mispriming from other than at their intended primer binding sites in a target nucleic acid.
Core Innovation
The invention is a method of amplifying a predetermined segment of a target nucleic acid by contacting a sample comprising the target nucleic acid with forward and reverse primers and conducting an amplification reaction in which an amplification product is formed by extension of the forward and reverse primers with the target nucleic acid serving as a template. The primers are designed so that one or more of the four standard nucleotide types are underrepresented, and the underrepresented nucleotide type(s) are the same in the primers.
The underrepresented nucleotide type(s) in each primer are present at two or fewer internal positions and/or at the 5′ end position. The primers are not random primers in which most or all primer positions are occupied by degenerate selections of nucleotides varying between primers, and the method is performed in multiplex with one or more further pairs of forward and reverse primers forming amplification products of further predetermined segments, with the underrepresented nucleotide type(s) being the same for each of the further pairs.
In multiplex, for each primer of each further pair, the underrepresented nucleotide type(s) are present at two or fewer internal positions and/or at the 5′ end position. The limited nucleotide composition of the primers suppresses primer-dimer and nonspecific amplification by limiting primer-primer interaction availability and pairing constraints, including cases involving short primer binding sites and optional helper structures when binding sites are short.
The approach is described as compatible with multiple multiplex amplification formats and with a broad set of detection and monitoring strategies, including SYBR intercalating dye with melt-curve multiplexing, fluorescence/quencher formats, and labeled detection schemes involving cleavage or universal tail and probe readouts. Documented use cases include sequencing library preparation, mutation detection, immunoPCR, whole genome amplification, and diagnostic and genomic analysis kit/applications.
Claims Coverage
Independent claim clm-00001 defines a multiplex amplification method using primers that share the same underrepresented nucleotide type(s) and restrict the underrepresented nucleotide type(s) to a limited number of positions, while excluding random degenerate primers. Main inventive features include constrained underrepresented nucleotide composition in the primers and extension-based amplification in multiplex with further primer pairs sharing the same underrepresented nucleotide type(s).
Underrepresented nucleotide composition primers for extension amplification
A method of amplifying a predetermined segment by contacting a sample with forward and reverse primers and conducting an amplification reaction forming an amplification product by extension of the primers with the target nucleic acid as template, wherein the primers are underrepresented in one or more of the four standard nucleotide types and the underrepresented nucleotide type(s) are the same in the primers.
Limited position constraint and exclusion of random degenerate primers
The underrepresented nucleotide type(s) are present at two or fewer internal positions and/or the 5′ end position in each primer, and the primers are not random primers in which most or all primer positions are occupied by degenerate selections of nucleotides varying between the primers.
Multiplex amplification with further primer pairs sharing the same underrepresented nucleotide type(s)
The method is performed in multiplex with one or more further pairs of forward and reverse primers forming amplification products of one or more further predetermined segments, wherein the underrepresented nucleotide type(s) are the same for each of the further pairs and, in each primer of each further pair, the underrepresented nucleotide type(s) are present at two or fewer internal positions and/or at the 5′ end position.
The claim coverage centers on constrained primer design using a shared underrepresented nucleotide type(s) limited to two or fewer internal positions and/or at the 5′ end position, applied both to the primer pair for a predetermined segment and to additional primer pairs in multiplex, while excluding random degenerate primers.
Stated Advantages
Suppresses primer-dimer and nonspecific amplification.
Improves detection in the constrained primer design context.
Documented Applications
Sequencing library preparation.
Mutation detection.
ImmunoPCR.
Whole genome amplification.
Diagnostic and genomic analysis kit/applications.
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